Upon completion of the prototype self-referencing polarographic (Serp) system for the detection of cellular oxygen flux, the groups' effort has shifted to testing and refinement. There has also been considerable work done on understanding the analysis of the resulting data. Two types of testing have been completed using either artificial or biological sources of oxygen concentration gradients. Within the oxygen gradient the difference in oxygen concentration between two points can be used to determine oxygen flux based on Fick's law. While this is straight forward, understanding the steady-state dynamics of oxygen concentration within a gradient around a glass micropipette acting as a source oxygen, has proved more difficult. The results are that we better understand this system and can now apply this knowledge to other Serp-electrode techniques. The results of the testing of the Serp-oxygen electrodes in this artificial environment have shown that the system reliably, and accurately, measures oxygen flux. Furthermore, the sensitivity of the system has been much improved by a recent upgrade of the main amplifier. This improved instrumentation is good enough to allow spatial resolution of oxygen flux associated with changes in mitochondrial localization within a single cell. We have been using this new technology to measure oxygen flux around a variety of different types of single cells. The addition of temperature control capabilities to the Serp system has allowed us to measure oxygen flux in a variety of mammalian cells at normal physiological temperatures. Specifically, studies have been completed that examined metabolic oxygen consumption by mouse oocytes and developing embryos. These studies have allowed us to demonstrate that the electrodes are robust enough, despite their delicate nature, to operate at temperatures as high as 37o C. This is of paramount importance if the system is to be reliably applied to biomedical research. Clearly this technique has passed through the prototype stage and is now a significant resource to investigators at the Center. We are already witnessing interest and applications to use this technology from biomedical researchers. Given that the system and supporting electronics have been perfected, we can now shift our focus to the development of new microelectrode construction techniques that will allow us to build improved oxygen microelectrodes, as well as beginning the development of the Serp-nitric oxide detection system.